Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin.
Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.
Lazarowski
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Division of Cell Biology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Lapetina
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EG
D001792:Blood Platelets / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006801:Humans; D007295:Inositol Phosphates / Q000097:blood / Q000302:isolation & purification; D007700:Kinetics; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000097:blood*
D007295:Inositol Phosphates; D010738:Type C Phospholipases; D014357:Trypsin; D013917:Thrombin